Kawasaki disease (KD) is an acute febrile vasculitis syndrome which occurs throughout the body, and the etiology of kawasaki disease remains unknown. The disease was first discovered by a Japanese scholar Tomisaku Kawasaki in Japan in 1961, and was first reported in 1967. Since 1970, kawasaki disease has been successively reported in almost all countries and regions in the world, and the highest incidence is in Asian. Recently, KD often occurs in children under five years of age. KD is characterized by fever, mucositis, rash, cervical lymphadenopathy and changes in the extremities. The pathological features of KD mainly include systemic vasculitis involving small- and medium-sized artery vascular, especially, KD can cause inflammatory injury in coronary arteries, which would lead to thrombotic infarction, stenosis, ectasia and aneurysms. Giant coronary aneurysm may occur in some children patient, and the long-standing giant coronary aneurysm can cause coronary artery stenosis or occlusion in the later stage, which may lead to ischemic heart disease and even death. In addition, KD can also lead to myocardial hypertrophy, focal myocardial ischemia, myocardial fibrosis, myocardial infarction in adulthood, and even sudden death in severe cases. Even KD patients with coronary artery aneurysms timely receive targeted and effective treatment, some patients will still die due to giant coronary artery aneurysms. The incidence rate of coronary artery ectasia has been reported to be 18.6-26.0%, and the incidence rate of coronary artery aneurysms has been reported to be 3.1%-5.2%. What's more, the incidence rate of coronary artery aneurysms has been reported to be on the rise year by year. Coronary artery aneurysm is the most serious complication of KD. When coronary artery aneurysm occurs, the patient is at high risk for developing vascular intima thrombus and hyperplasia, which may result in stenosis adjacent to the lumen of coronary artery. The retaining blood in the aneurysm is at high risk for developing thrombus, which will reduce blood flow to the region adjacent to the coronary artery aneurysm and cause myocardial infarction and sudden death. When the diameter of the aneurysm is ≧8 mm, the aneurysm are classified into giant aneurysm, its regression will be more difficult, and the incidence rate of stenosis will significantly increase as time goes on. In a word, KD puts children patient at high risk and brings serious impact on their lives. At present, the treatment methods for coronary artery aneurysms caused by KD include long-term anticoagulant therapy, thrombolytic therapy, coronary artery bypass surgery, heart transplantation and interventional therapy. However, all of these methods are remedial therapies, and the results are usually not satisfactory. The cost of treatment is considerably high, and quality of life in treated children can't be guaranteed.
It has been reported that in developed countries or regions, such as Japan and the United States, the coronary complication caused by KD has replaced rheumatic fever as the most common cause of acquired heart disease in children, and is one of dominant factors for ischemic heart disease in late adulthood. In China, the incidence rate of KD is also high, next only to Japan and South Korea, and has already replaced rheumatic fever as the most common cause of acquired heart disease in children. What′ more, its incidence in China is increasing in recent years, which poses a potential health risk to children's heart and vascular and great economic burden to society.
At present, KD can be diagnosed by clinical symptoms, ultrasound imaging and laboratory tests. The clinical diagnosis of KD is mainly based on clinical symptoms, however, children are often not diagnosed until they have been found several typical clinical symptoms and other illnesses with similar symptoms have been ruled out. Therefore, the timeliness of clinical diagnosis is poor. For ultrasound imaging method, the pathological changes in coronary artery are determined by echocardiography images, however, the ultrasound imaging method has its limitation for diagnosis of early or minor coronary artery disease in children with KD. For laboratory tests, the systemic inflammatory indexes are used to facilitate the diagnosis of KD in children, including increased peripheral blood leukocytes and neutrophils in the acute phase, mild anemia, increased platelets, significantly increased C-reactive protein, and significantly increased rythrocyte sedimentation rate, etc. The inflammatory indexes can only be seen as assisted or indirect diagnosis references, so the specificity and pertinence of the laboratory tests are not ideal. What's more, many other diseases, especially infectious diseases, may also cause systemic inflammation, so the common diagnostic methods often result in confusions with other infectious diseases, which then lead to delayed treatment.
At present, the clinical diagnosis of atypical KD is relatively difficult. The incidence rate of atypical KD is approximately 10%-36%, and is increasing year by year. The clinical symptoms of atypical KD are also diverse and complex, and it is more likely to be misdiagnosed as respiratory tract infection, sepsis, drug rash, scarlet fever, measles, lymph node inflammation, juvenile rheumatoid and other diseases. Patients often miss optimal timing for treatment due to misdiagnosis or missed diagnosis. As a result, coronary artery injuries have already occurred in many children when they are definitely diagnosed with KD.
Recently, scholars have been working hard to find markers for early diagnosis of KD. However, since the etiology and pathogenesis of KD remain unknown, most scholars focus on genes, cytokines, and inflammatory cytokines, the biomarkers that have been found can't be used as specific markers for diagnosis of KD. Although it has been reported that some proteins and genes, such as heart-type fatty acid binding protein (h-FABP), matrix metalloproteinase-9 (MMP-9), and N-terminal B-type natriuretic peptide (NT-pro BNP), could be used as molecular markers for diagnosis of KD, the molecular markers are unable to satisfy both sensitivity and specificity, and are more likely to cause detection error resulting from sampling methods and operating procedure, etc., thus, the molecular markers have not been confirmed by large-scale clinical trials. To date, there is no universally recognized marker and methods for diagnosis of KD. Therefore, it is important to find a molecular marker for rapid and accurate diagnosis of kawasaki disease, which is useful for the clinical treatment of KD so as to avoid occurrence of coronary artery disease, improve prognosis and quality of life in treated children.
Exosomes are cell-secreted vesicles derived from late endosomes (also known as multivesicular endosomes). The internal vesicles are released into the extracellular space when the multivesicular endosomes fuse with the plasma membrane. It has been reported that exosomes derived from different cells contain key functional molecular constituents of their cells of origin. The reported diameter of exosomes is between 30 and 100 nm. Exosomes are present in many cells, and contain various molecular constituents, including proteins, lipids and micro RNAs. The exosomal proteins and microRNAs vary with the cells and tissues of origin, as well as the biological function of the exosomes. Exosomes in blood are solid components with low density and are reported to contain a great deal of biomarker information, thus exosomes have attracted widespread attention in recent years. In human body fluids (such as serum, urine, tissue fluid), RNAs wrapped in exosomes will not be degraded by nuclease, and will not be affected by highly expressed proteins (such as albumin, IgG). The constituents contained in exosomes, which are part of constituents of their cells of origin, give a possibility to detect changes in certain proteins and nucleic acids in the cell. In recent years, increased attention has been paid to the role that exosomes in body fluids may play in clinical diagnosis. For example, micro RNAs present in serum of cancer patients can be used as molecular markers for early diagnosis of several types of cancers. In addition, OMICS provides an optimal platform and technique to detect specific molecular markers for diseases of unknown etiology. OMICS informally refers to a field of genomics, proteomics, transcriptomics, etc., OMICS aims at the collective characterization and quantification of all DNA, RNA or proteins in samples by certain experiments and data analysis. It is expected to detect disease-specific molecular markers by comparing data from normal individual and data from patient, and the molecular markers may play an important role in early diagnosis, etiological analysis, in-depth study and treatment of disease, etc. Currently, these methods have been widely used in the studies of cell proliferation, differentiation, abnormal transformation, tumor formation and other aspects, involving liver cancer, breast cancer, colon cancer, bladder cancer, prostate cancer, lung cancer, kidney cancer, neuroblastoma, etc. A series of tumor-associated proteins have been identified, which is helpful for early diagnosis of tumor, detection of drug targets, therapeutic evaluation and prognosis. Some nucleic acid molecules can also be used as molecular markers for diagnosis of diseases. The nucleic acid markers have high sensitivity and good specificity in clinical diagnosis, and can be accurately quantified, thus they are especially suitable as early diagnostic markers.
Mature microRNA (miRNA) is a small non-coding RNA molecule with sizes of 17-25 nucleotides. MicroRNA inhibits the translation of target mRNA via base-pairing with complementary sequences within 3′-UTR, 5′-UTR and encoding region of target mRNA, that is to say, miRNA functions in post-transcriptional regulation of target gene expression. Bioinformatics studies show that each miRNA can regulate multiple target genes, whereas one target gene can also be regulated by multiple miRNAs. According to conservative estimates, miRNAs appear to target about 60-70% of human protein encoding genes. A single miRNA may bind with hundreds of target mRNAs with different functions, so as to play regulatory roles. MicroRNAs involve in almost all physiological and pathological processes in mammalian, such as ontogenesis, tissue differentiation, apoptosis and energy metabolism, thus miRNAs are associated with occurrence and development of various diseases.
The previous studies on miRNAs mainly focus on their actions in cells. In 2008, Mitchell et al. constructed a small RNA library by isolating RNAs with sizes of 18-24 nucleotides from plasma of healthy people, 125 DNA clones obtained were sequenced and analyzed, and 37 miRNA molecules in the plasma samples were cloned, including let-7a, miR-16, miR-15b, etc. It is found that miRNAs can exist in human plasma in a stable form, which prevents miRNAs from degradation by endogenous RNase. During the same time period, Chen et al. analyzed miRNAs in serum by high-throughput sequencing technology, and more than 100 and 91 miRNAs were detected in serum of healthy male and female people, respectively. It is found that these miRNAs remain stable even under extreme conditions (such as high temperature, extremely low or high pH, and multiple freeze-thaw), while most other RNAs have been degraded. In addition, according to the detection results of miRNAs in serum/plasma of healthy people and patients, miRNAs are found to widely exist in serum/plasma of healthy people and patient, and their expression profiles specifically vary with physiological status, disease types and disease degrees. Recent research shows that different tumors have specific miRNA profiles, and tumor-derived miRNAs can be released into circulatory system and blood tissue. MicroRNAs in blood can't be degraded by RNase and thus are quite stable. Therefore, miRNAs in serum or plasma have potential as cancer biomarker. For example, recent research finds that the expression level of miR-21 in serum of patients with diffuse large B-cell lymphoma is higher than that in serum of healthy people. Since there are plenty of stable microRNAs in human serum or plasma, and exosomes in blood are reported to carry abundant biomarkers information, and exosomes derived from different cells contain key functional moleculars of their cells of origin, increased attention has been paid to the role that exosomes in body fluids may play in clinical diagnosis. For example, microRNA molecular markers in the serum of cancer patient have been used for early diagnosis of various cancers. Based on the above phenomena, we could detect exosomal microRNAs whose expression levels change obviously in the serum of patient with KD, and then use these exosomal microRNAs as biomarkers for early diagnosis of KD.